Although a two-dimensional (2D) cell culture model achieves recognition of the value in the biomedical science research, because the 2D cell culture model is performed using a petri dish to 2-dimensionally culture cells on a bottom of the petri dish, many cell types of tissue-specific and differentiated functions are not described or tissue functions and drug activity in vivo are not exactly predicted.
Particularly, in case of brain cells having a neuron and a neural stem cell which 3-dimensionally contact each other in vivo, owing to a limitation of the 2D cell culture model, an interest in a 3D cell culture model which simulates a spatial structure and biochemical complexity of living cells well has been increased.
The 3D cell culture model is performed, due to imitating a situation in vivo well, to implement a directional growth and complexity of cell-cell binding in an experiment in vitro. Further, the 3D cell culture model shows an improved cell survival and an enhanced neuronal differentiation as compared with the 2D cell culture model.
Accordingly, the 3D cell culture model is useful for identifying signaling pathways and drug responsiveness of various disease states well as compared with the 2D cell culture model in researching the molecular basis tissue function.
Meanwhile, for example, the 3D cell culture model was disclosed in an article (see the detailed description of the following ‘related art’) with a title of ‘Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor’.
The article proposed the 3D cell culture model which simulates a blood-brain barrier (BBB) by using a neurovascular microfluidic bioreactor in vitro. Herein, the BBB is composed of endothelial cells, pericytes, and astrocytes and functions as a gatekeeper limiting the transport of materials between a blood and a brain tissue.
That is, the BBB serves to send nutrients in the blood required to the brain tissue toward the brain from the blood and prohibit the entry of potentially harmful substances from a blood stream into the brain tissue. Herein, the neurovascular microfluidic bioreactor is configured to operate by using a first to a third polydimethylsiloxane (PDMS) layers which are sequentially laminated on a glass and a polycarbonate membrane between the first and second PDMS layers.
The membrane is a semi-permeable membrane and sets up a borderline between a microfluidic vasculature and brain compartments in the neurovascular microfluidic bioreactor. The first PDMS layer has two perfusion ports for vascular media supply in the endothelial cell and the second and third PDMS layers have four perfusion ports for brain media supply.
In order to grow cells at both sides of the membrane, first of all, the first PDMS layer is positioned at a higher level than the second and third PDMS layers and perfused through two perfusion ports in order to seed the endothelial cell in a conduit side of the membrane.
Next, after the first PDMS layer is positioned at a lower level than the second and third PDMS layers, the second and third PDMS layers are perfused through four perfusion ports in order to seed the neuron, the astrocyte, and the pericyte included in collagen in a brain side of the membrane.
Next, the endothelial cell, the neuron, the astrocyte, and the pericyte are cultured through a cell culture medium on the first and second PDMS layers to form the BBB around the membrane. However, the neurovascular microfluidic bioreactor is required to be reversed with respect to the first PDMS layer while forming the BBB, and a neurovascular unit (NVU) in vivo including the BBB, which are composed of the endothelial cell, the astrocyte and the pericyte, is not sufficiently implemented.
The reason is that the NVU has the endothelial cell positioned at the conduit side of the membrane and the astrocyte, the pericyte and the neuron positioned at the brain side of the membrane, which are separated by the membrane to be incompletely simulated from the viewpoint of a structure of the BBB in vivo, and also, other brain cells other than the endothelial cell, the pericyte, the neuron, and the astrocyte are further required from the viewpoint of the BBB in vivo and a structure therearound.
Further, the neurovascular microfluidic bioreactor 2-dimensionally cultures the endothelial cell along the membrane on the first PDMS layer to prevent the BBB in vivo from being sufficiently implemented and contacts the endothelial cell and the cell culture medium on the PDMS layer having a characteristic of absorbing air or drugs to show a different aspect from a mechanism in vivo related with the BBB.
Accordingly, because a simulation technique of the NVU in vitro which is developed in the related art shows the different aspect from the mechanism in vivo and has a problem in that efficacy of candidate drugs and predictability for the toxicity are low, for researches of the brain diseases, new drug development of the brain diseases, and patient-specific personalized treatment, development of an NVU on-a-chip having high degree of simulation is required.